Research

Overview

The goal of the research in the Lopes lab is to understand the transcriptional
regulation of phospholipid biosynthesis in yeast, and how phospholipid biosynthesis is
coordinated with other biological processes. To address this goal, we are carrying on two
inter-related projects. One project is designed to determine how phospholipid
biosynthesis is coordinated with other biological processes via a set of transcription
factors that belong to the basic helix-loop-helix (bHLH) family. Another project is
focused on the regulation of the only essential phospholipid biosynthetic gene (PIS1)
required for the synthesis of phosphatidylinositol (PI). This project is driven by the fact
that virtually nothing is known about the transcriptional regulation of the PIS1 gene
except that it is not coordinated with expression of the other phospholipid biosynthetic
genes.

Regulation of yeast phospholipid biosynthesis requires the Ino2p:Ino4p activators,
which belong to the bHLH family of transcription factors. Initially, we were
focused on the regulation of the INO2 and INO4 genes and found that INO2 expression is
auto-regulated in a pattern that is identical to the regulation of the Ino2p target genes.
More recently, we also carried out genetic analyses of these genes to define their roles in
transcriptional regulation.

Currently, we have turned our attention to
how phospholipid biosynthesis is coordinated
with other biological processes via the bHLH
proteins. Yeast has nine bHLH proteins that
regulate several different processes including:
phosphate utilization, retrograde response
(control of nuclear genes in response to
mitochondrial damage), and glycolysis (Fig.
1). Since bHLH proteins are known to form
multiple dimer combinations in other
organisms, we reasoned that dimer partner
selection could be a mechanism for
coordination of the biological processes
regulated by yeast bHLH proteins. Thus, we
defined the yeast bHLH protein interaction map using the yeast two-hybrid system and
co-purification of epitope-tagged recombinant proteins. As we predicted, this map
identifies several novel bHLH dimer combinations (Fig. 1). In addition, since specific
growth conditions affect expression of specific bHLH genes, then different growth
conditions would be expected to change the bHLH protein interaction map. We are
presently testing this prediction.

We are also determining the role of the yeast bHLH proteins in coordination of
different biological processes using well-characterized candidate target genes for each biological process. To complement the candidate gene approach, microarray analysis is
being used to define the target genes for each bHLH protein under conditions that affect
the expression of the different bHLH encoding genes. Lastly, to define the mechanism(s)
whereby bHLH dimers coordinate gene expression, we are determining the autoregulatory
circuits that control expression of each bHLH-encoding gene.

Genomic analysis of PI synthesis in yeast

Fig. 2. PI metabolism pathways with associated cellular functions.

Recently, we have also focused our attention on the regulation of PIS1 expression
The PIS1 gene is required for the synthesis of PI. PI and its metabolites (inositol
polyphosphates, phosphoinositides, and sphingolipids) play important roles in a myriad
of processes, including: chromatin remodeling, export of mRNA from the nucleus,
vesicle trafficking, and signal transduction (Fig. 2). In spite of the importance of PI to
the cell, relatively little is
known about how its levels
are regulated, particularly at
the level of transcription of
the PIS1 gene. Promoter
deletion analysis reveals
that the PIS1 promoter
includes five regulatory
elements: three upstream
activation sequences (UAS)
and two upstream repression
sequences (URS). Our
published studies reveal the
carbon source regulates
PIS1 expression through the
URSGLY element and
probably via a novel
regulatory cascade. We
have also determined that oxygen regulates PIS1 expression through the Rox1p repressor
protein and the URSROX element. Screening a yeast gene deletion set, we identified 120
genes that affect expression of a PIS1 reporter. These genes suggest that processes such
as peroxisomal function and DNA repair are coordinated with PI synthesis.

The knowledge that PI metabolites have roles that affect gene expression suggests
that regulation of PI synthesis may affect several cellular processes. We are testing this
directly by determining the phenotype of strains with altered PI synthesis and by using
microarray hybridization to identify genes that are regulated in response to PI levels.